MICRORNA501 UP-REGULATION MAY INCREASE THE AGGRESSIVENESS OF CLEAR CELL RENAL CARCINOMA THROUGH MTOR ACTIVATION AND P53 DEGRADATION

Introduction: Many biological processes as gene expression, cell proliferation, differentiation and apoptosis are affected by microRNAs (miR) which are small noncoding RNAs that act at post transcriptional level. In fact, the impaired function of these short RNAs might cause several diseases including cancer (1). We have found a variable expression of miR501 in 63 pairs of normal and clear cell renal carcinoma (ccRCC) tissues, therefore, a possible function of this miR in ccRCC was investigated. Material and Methods: Analysis of miR501 expression was carried out by microarray and real time RT-PCR. MiR501 up or downregulation was performed by cell transfection with a specific plasmid expressing miR501 sequences (PL-501) and antagomiR, respectively. Apoptosis was analysed through caspase-3 activity, Hoechst method and cell cycle analysis. Cell proliferation was evaluated by direct cell count and with the CellTiter method. Gene expression, protein ubiquitination and kinase activity were analysed by immunological techniques and cell imaging. Results: Follow up analysis of 35 ccRCC subjects showed a good prognosis for patients which a lower expression of miR501 in ccRCC tissues compared with normal renal parenchyma. Conversely, about the 50% of patients with unchanged or higher levels of miR501 exhibited a poor prognosis. In order to evaluate the role of miR501 in renal cancer, we have modified its expression transfecting kidney carcinoma cells KJ29 (2) with a specific antagomiR and with the PL-501 plasmid. MiR501 downregulation caused a reduction of mTOR activity, the increase of G0/G1 phase of cell cycle and induced apoptosis by enhancing the activity of caspase-3. Activation of apoptosis occurred in a p53-dependent manner without affecting the expression of the mTOR-related MDM2 protein, an inhibitor of p53, which results overexpressed in metastatic kidney carcinoma (3). On the other hand, miR501 upregulation caused mTOR activation that stimulated cell proliferation as well as cell survival. The latter biological processes were associated with an increased expression of MDM2 which induced p53 degradation activating the proteasome by p53 poly-ubiquitination. Discussion and conclusion: MiR501 seems to act as a molecular switch able to turn on or off mTOR signalling. In fact, the downregulation of miR501 led to sequential mTOR kinase inhibition, p53 activation and increased apoptosis. Conversely, miR501 upregulation caused mTOR activation, increased expression of MDM2 and p53 degradation, promoting cell survival, as already observed for follow up data. These findings support the role of kingmaker for the miR501 among apoptosis and cell survival in ccRCC patients, therefore miR501 expression could be used to evaluate the prognosis of patients with clear cell renal carcinoma. 1. Di Leva G, Croce CM: Roles of small RNAs in tumor formation. Trends Mol Med. 16(6):257-67, 2010. Review. 2. Barletta C et al.: Cytogenetic, molecular and phenotypic characterization of the newly established renal carcinoma cell line KJ29. Evidence of translocations for chromosomes 1 and 3. Anticancer Res. 15(5B):2129-36, 1995. 3. Noon AP et al: Combined p53 and MDM2 biomarker analysis shows a unique pattern of expression associated with poor prognosis in patients with renal cell carcinoma undergoing radical nephrectomy. BJU Int. 109(8):1250-7, 2012.